CD8 T cells play a crucial role in defense against viral infections like HIV, hepatitis B and C, Epstein Barr virus, cytomegalovirus, and arenaviruses. A major drive therefore has been to develop novel vaccination strategies that can elicit potent CD8 T cell memory and engender effective protective immunity against these viral pathogens. Recent studies have indicated that memory CD8 T cell precursors are present as a subset of effector cells at the peak of the primary T cell response. However, unlike competent memory cells, these memory precursors are in a state of cell cycle arrest; they lack the ability to self renew by homeostatic proliferation or proliferate in response to antigen re-call, and provide poor protective immunity. These memory precursors embark on a program of differentiation to become competent memory cells by a process lasting up to several weeks after antigen clearance. Despite the recognition that effector to memory differentiation is a rate-limiting step in the development of CD8 T cell-dependent protective immunity, the mechanisms underlying the induction and subsequent reversal of proliferative block in memory precursors are poorly defined. Our studies using the mouse model of lymphocytic choriomeningitis virus (LCMV) have shown that genetic deficiency of cyclin dependent kinase inhibitor p27Kip1 not only accelerated the differentiation of memory cells from effector CD8 T cells, but also significantly increased the magnitude of CD8 T cell memory. Based on these findings, our central hypothesis is: p27Kip1 impedes differentiation of memory CD8 T cells from primary effector CD8 T cells by opposing cell cycle re-entry in response to mitogenic cues. We further hypothesize that memory CD8 T cell differentiation including p27Kip1 expression is regulated by the upstream FOXO transcription factors. This proposal focuses on elucidating the key roles of FOXO/p27Kip1 pathway in regulating the differentiation of memory CD8 T cells through these specific aims: 1) To test the hypothesis that effects of p27Kip1 in regulating memory cell differentiation are CD8 T-cell autonomous 2) To test the hypothesis that FOXO transcription factors regulate differentiation of memory CD8 T cells. Aims 1 and 2 will utilize complementary approaches including TCR transgenic (tg) mice, bone marrow chimeras, mice that are conditionally deficient for p27Kip1 or FOXOs in T cells, and inducible p27Kip1/FOXO tg mice to determine the in vivo role of p27Kip1 and FOXOs in regulating the dynamics of phenotypic and functional modifications that occur in CD8 T cells during differentiation from effectors to competent memory cells. These studies would provide critical insight into the mechanisms governing memory cell differentiation, and also have the potential to identify p27Kip1 and FOXO as molecular targets for development of novel immunotherapies to accelerate and enhance the induction of vaccine-induced CD8 T cell-dependent protective immunity against viral infections. PUBLIC HEALTH RELEVENCE: CD8 T cells play a critical role in defense against several viral infections of humans. In this proposal, we seek to identify key molecular regulators (p27Kip1 and FOXO) that can be modulated in CD8 T cells by immunotherapies to enhance vaccine-induced protective anti-viral immunity. These studies should aid in the development of effective vaccines to protect humans against virally induced diseases.